Abstract

Background. Recent studies have shown that renal expression of 25-hydroxyvitamin D-3-1 alpha -hydroxylase (1 alpha -OHase) is not restricted to proximal tubules. To investigate the significance of this expression, we characterized the regulation of 1 alpha -OHase expression and activity in a human cortical collecting duct cell line (HCD).

Methods. Expression of 1 alpha -OHase mRNA and protein was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Enzyme activity was quantified using 25-hydroxyvitamin D-3 as the substrate; conversion to 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] and 24,25-dihydroxyvitamin D-3 was then determined by thin-layer chromatography.

Results. HCD cells expressed mRNA and protein for 1 alpha -OHase. However, basal 1,25(OH)(2)D-3 production was lower than that observed in proximal tubule HKC-8 cells. In both cell lines, synthesis of 1,25(OH)(2)D-3 was increased by forskolin, parathyroid hormone, and low calcium medium. Conversely, treatment with 1,25(OH)(2)D-3 itself decreased 1 alpha -OHase activity. This effect was more pronounced in HCD cells, which also demonstrated significantly higher levels of 24-hydroxylase activity. The most striking induction of 1 alpha -OHase activity was observed in the HCD cells following incubation with lipopolysaccharide. which was coincident with the expression of mRNA for both CD14 and Toll-like receptor 4.

Conclusions. These results highlight the capacity for synthesis of 1.25(OH)(2)D-3 in cells from more distal areas of the nephron. However, more sensitive feedback regulation and immune induction of 1 alpha -OHase in the HCD cells suggest a more localized role for 1,25(OH)(2)D-3 production in the distal nephron.